BRCA1 and BRCA2 are tumor suppression genes shown to be involved in 90% of familial breast and ovarian cancers (Miki et al. Science (1994) 266, 66-71). The full length gene sequence of BRCA1 is disclosed by Miki et al. and depicted herein as SEQ ID NO:1. The full length gene sequence of BRCA2 is deposited in GenBank, Accession Number U43746. Mutations in the breast and ovarian cancer susceptibility gene, BRCA1, account for about half of the inherited breast and ovarian cancers (Miki et al. (1994) Science, 266, 66-71; Easton et al. (1995) Am. J. Hum. Genet., 56, 265-271; Ford et al. (1995) Am. J. Hum. Genet., 57, 1457-1462) and about 10% of the sporadic ovarian cancers (Futreal et al. (1994) Science, 266, 12-122; Hosking et al. (1995) Nature Genet., 9, 343-344; Merajver et al. (1995) Nature Genet., 9, 439-443). BRCA2 was found to be associated more frequently with male breast cancer compared to BRCA1 (Wooster, R. et al. 1994. Nature 265:2088-2090. Patients with BRCA2 mutations were also found to be at higher risk for a variety of other cancers including carcinomas of the pancreas, prostate, and colon (Thorlacius, S. et al. 1996. Nat. Genet. 13:117-119; Phelan, C. M. et al. 1996. Nat. Genet. 13:120-122; Gudmundsson, J. et al. 1995. Cancer Res. 55:4830-4832; Tonin, P. et al. 1995. J. Med. Genet. 32:982-984).
The BRCA1 cDNA codes for a 1863 amino acid protein with an amino terminal zinc ring finger domain and a carboxy terminal acidic region (Miki et al. 1994 Science, 266, 66-71) typical of several transcriptional factors. The BRCA2 gene is composed of 27 exons and encodes a protein of 3418 amino acids with no significant homology to any known protein (Wooster, R. et al. 1995. Nature 378:789-792; Bork, P. et al. 1996. Nat. Genet. 13:22-23). Recently, the C terminal region of BRCA1 was shown to activate transcription in a heterologous GAL-4 system (Chapman, M. S. and Verma, I. M. (1996) Nature, 382, 678-679; Monteiro et al. (1996) Proc. Natl. Acad. Sci. USA, 93, 13595-13599). Murine BRCA1 has been cloned and the developmental patterns of expression studied (Lane et al. (1995) Genes and Development, 9, 2712-2722; Marquis et al. (1995) Nature Genetics, 11 17-26; Abel et al. (1995) Hum. Molec. Genet., 4, 2265-2273; Sharan et al. (1995) Hum. Mol. Gen., 4, 2275-2278). Expression was found to be high in rapidly proliferating tissues (Lane et al. (1995) Genes and Development, 9, 2712-2722; Marquis et al. (1995) Nature Genetics, 11 17-26), particularly those undergoing differentiation, thus suggesting a role for BRCA1 in cellular growth and differentiation. The BRCA1 gene product has been shown to be a nuclear phosphoprotein (Chen et al. (1995) Science, 270, 789-791; Rao et al. (1996) Oncogene, 12, 523-528; Scully et al. (1996) Science, 272, 123-125) that, when overexpressed in breast and ovarian cancer cells, results in growth inhibition in vitro and in vivo. Conversely, inhibition of BRCA1 expression by antisense RNA in mouse fibroblasts or by antisense oligonucleotides in breast cancer cells resulted in transformation of mouse fibroblasts as well as an increase in the rate of growth of breast cancer cells (Thompson et al. (1995) Nature Genetics, 9, 444-450; Rao et al. (1996) Oncogene, 12, 523-528).
Two new alternately spliced BRCA1 transcripts referred to as BRCA1a and BRCA1b have recently been isolated and mouse fibroblast cell lines and human breast cancer cell lines expressing BRCA1a proteins have been developed (Shao et al. (1996) Oncogene, 13, 1-7). Overexpression of BRCA1a was found to induce apoptosis in NIH3T3 and MCF-7 cells after calcium ionophore treatment thus indicating that BRCA1 proteins, specifically BRCA1a, play a role in the regulation of apoptosis (Shao et al. (1996) Oncogene, 13, 1-7). The role of BRCA2 in apoptosis remains to be elucidated.
Two proteins, BARD1 and Rad51 which are human homologs of bacterial Rec A, were shown to interact both in vitro and in vivo with BRCA1 and BRCA2 indicating a role for BRCA1 proteins in tumor suppression and a role for BRCA1 in the control of recombination and genomic integrity, as well as a role for BRCA2 in DNA repair (Wu et al. (1996) Nat. Genetics, 14, 430-447; Scully et al. (1997) Cell, 88, 265-275; Sharan, S. K. et al. 1997. Nature 386:804-810; Zhang, H- T. et al. 1998. Cell. 92:433-436).
Previously, the BRCA1 gene product was shown to be localized in the nucleus (Chen et al. (1995) Science, 270, 789-791; Rao et al. (1996) Oncogene, 12, 523-528). However, differences regarding the size and subcellular localization of BRCA1 have been reported (Chen et al. (1995) Science, 270, 789-791; Chen et al. (1996) Science, 272, 125-126; Jensen et al. (1996) Nature Genetics, 12, 303-308; Scully et al. (1996) Science, 272, 123-125; Thakur et al. (1997) Mol. Cell. Biol., 17, 444-452; Wilson et al. (1997) Oncogene, 14, 1-16). Two additional BRCA1 splice variants, BRCA1xcex94672-4092 (which lacks exon 11) and BRCA1xcex9411b (which lacks a majority of exon 11) were recently found to localize to the cytoplasm by immunostaining (Thakur et al. (1997) Mol. Cell. Biol., 17, 444-452; Wilson et al. (1997) Oncogene, 14, 1-16). BRCA1xcex9411b was also found to be present in significant quantities in the nuclear fractions on immunoblotting analysis.
Both BRCA1 and BRCA2 gene products have been reported to be regulated in a cell cycle-dependent manner and to have a potential transactivation function (Rajan, J. V. et al. 1996. Proc. Natl. Acad. Sci. USA 93:13078-13083; Vaughn et al. (1996) Cancer Res. 56, 4590-4594; Chapman, M. S. and I. M. Verma. 1996. Nature 382:678-679; Monteiro, N. A. et al. 1996. Proc. Natl. Acad. Sci. USA 93:13595-13599; Milner, J. et al. 1997. Nature 386:772-773; Cui, J. et al. 1998 Oncology Reports 5:585-589).
The sequences of BRCA1a and BRCA1b, two splice variants of BRCA1, have now been determined. It has been found that BRCA1a lacks a majority of exon 11 (amino acids 263-1365) while and BRCA1b lacks exons 9, 10 and a majority of exon 11 (amino acids 263-1365). Like BRCA1, BRCA1a encodes a phosphoprotein containing phosphotyrosine that associates via its amino-terminal zinc ring finger domain with E2F transcriptional factors, cyclin and cyclin dependent kinase (cdk) complexes. The amino-terminal region of BRCA1a has now been demonstrated to function as a transactivation domain when fused to heterologous GAL4 DNA binding domain. Additional studies indicate the presence of a negative regulatory domain at the carboxy-terminal regions of BRCA1 and BRCA1a proteins. It is believed that mutations in the zinc ring domain found in patients with breast and ovarian cancer may impair this activity, thus indicating that a loss of transcriptional activation by BRCA1 may lead to the development of breast and ovarian cancers.
The protein encoded by the BRCA1b splice variant, however, has now been found to have lost a portion of the amino-terminal transactivation domain as a result of alternate splicing. Accordingly, it is believed that BRCA1b may function as a dominant-negative regulator of the transcriptional activation function of BRCA1/BRCA1a proteins.
BRCA1 encoded proteins have now been found to accumulate in the cytoplasm in the presence of serum and in the nucleus in the absence of serum thus indicating that the nuclear localization of BRCA1 may be regulated by external stimuli, phosphorylation or proteinxe2x80x94protein interactions. Like BRCA1, proteins encoded by BRCA1a and BRCA1b both act as tumor suppressors, thus indicating that exon 11 is not need for this function. Proteins which interact with BRCA1 have also been identified.
It has also been found that the amino terminal region of BRCA2, like BRCA1, associates with transcriptional factor E2F, cyclin and cdk""s. BRCA2 has also been found to undergo differential splicing. The sequence of a BRCA2 splice variant, BRCA2a, has now been determined and contains a deletion of a putative transcriptional activation domain, giving rise to a protein with potential dominant negative pathophysiology. In addition, experiments examining the transcriptional factor function of BRCA2 demonstrate that the BRCA2 proteins have intrinsic histone acetyl transferase activity, activity that maps to the amino-terminal region of BRCA2. The BRCA2 proteins acetylate mainly H3 and H4 of free histones.